Interleukin-1 is a cytokine having a broad spectrum of biological activities (for reviews, see e.g., Dinarello, C. A. and Wolff, S. M. (1993) New Engl. J Med. 328:106-113; and Dinarello, C. A. (1993) Trends in Pharmacol. Sci. 4:155-159). IL-1 consists of two structurally related polypeptides, interleukin-1xcex1 (IL-1xcex1) and interleukin-1xcex2 (IL-1xcex2). The two forms of IL-1 are encoded by different genes and have only 27-33% amino acid identity but they interact with the same receptor and have similar biological activities. Included among the biological functions attributed to IL-1 are induction of fever, sleep, anorexia and hypotension. IL-1 is also involved in the pathophysiology of inflammatory and autoimmune diseases, including rheumatoid arthritis, septic shock, inflammatory bowel disease and insulin dependent diabetes mellitus. IL-1xcex1 has been specifically implicated in the pathophysiology of psoriasis. IL-1 is also thought to play a role in immune responses to infectious agents and in the pathogenesis of myeloid leukemias.
IL-1xcex1 and IL-1xcex2 are both synthesized as approximately 31 kDa precursor molecules that are subsequently processed to a mature form of approximately 17 kDa. IL-1xcex1 and IL-1xcex2 differ in that the precursor form of IL-1xcex1 (preIL-1xcex1) is biologically active and most of the mature IL-1xcex1 (matIL-1xcex1) remains cell-associated, whereas the precursor form of IL-1xcex2 (preIL-1xcex2) must be cleaved to its mature form to become active and the mature form of IL-1xcex2 (matIL-1xcex2) is secreted from the cell. Only certain cell types process preIL-1xcex2 and secrete matIL-1xcex2. Monocytes and macrophages are the most efficient producers and secretors ofIL-1xcex2, which is the most abundant form of IL-1 produced upon activation of these cell types.
Interleukin-1xcex2 converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1xcex2 cytokine from its inactive precursor (Black, R. A. et al. (1988) J Biol. Chem. 263:9437-9442; Kostura, M. J. et al. (1989) Proc. Natl. Acad. Sci. USA 86:5227-5231; Thornberry et al. (1992) Nature 356:768-774; Ceretti, D. P. et al. (1992) Science 256:97-100). ICE is a member of a family of cysteine proteases with shared homology. Other members of this family have been implicated in apoptosis, such as ced-3 (Yuan, J. et al. (1993) Cell 75:641-652), Nedd2 (Kumar, S. et al. (1992) Biochem. Biophys. Res. Commun. 185:1155-1161; Kumar, S. et al. (1994) Genes Dev. 8:1613-1626), CPP32 (Fernandes-Alnemri, T. et al. (1994) J. Biol. Chem. 269:30761-30764), Ich-1 (Wang, L. et al. (1994) Cell 78:739-750) and Ich-2 (Kamens, J. et al. (1995) J. Biol Chem. 270:15250-15256; Faucheu, C. et al. (1995) EMBO J. 14:1914-1922). The necessity for ICE in the generation of bioactive IL-1xcex2 was demonstrated in mice in which the ICE gene had been functionally disrupted (Li, P. et al. (1995) Cell 80:401-411. Kuida, K. et al. (1995) Science 26:2000-2003). Although these animals are overtly normal, they have a major defect in the production of mature IL-1-xcex2 after stimulation with lipopolysaccharide.
In vitro studies have demonstrated that ICE cleaves prointerleukin-1xcex2 at Asp116-Ala117 to release the fully active 17 kDa form (Black, supra; Kostura, supra). ICE also cleaves prointerleukin-1xcex2 at Asp27-Ala28 to release a 28 kDa form. Cleavage at these sites is dependent upon the presence of aspartic acid in the P1 position (Kostura, supra, Howard, A. et al. (1991) J. Immunol. 147:2964-2969; Griffin, P. R. et al. (1991) Int. J. Mass. Spectrom. Ion. Phys. 111:131-149). However, an aspartic acid in the P1 position is not sufficient for ICE specificity. For example, several other proteins containing Asp-X bonds, including prointerleukin-1xcex1, are not cleaved by ICE (Howard, supra).
ICE itself undergoes maturational processing, possibly performed in vivo by ICE itself (Thornberry, N. A. et al. (1992) Nature 3:768-774). Mature ICE is generated from a 404 amino acid precursor protein by proteolytic removal of two fragments, the N-terminal 119 amino acid xe2x80x9cpro-domainxe2x80x9d and the internal residues 298-316 (Thomberry, supra). Active ICE is therefore composed of two subunits, a 20 kDa subunit (p20) encompassing residues 120 to 297 and a 10 kDa subunit (p10) encompassing residues 317 to 404. The crystal structure of ICE indicates that ICE forms a tetrameric structure consisting of two p20 and two p10 subunits (Walker, N. P. C. et al. (1994), Cell 78:343-352; Wilson, K. P. et al. (1994) Nature 370:270-275). The catalytic amino acid residues of ICE are Cys-285 and His-237. The side chains of four amino acid residues (Arg-179, Gln-283, Arg-341 and Ser-347) form the P1 carboxylate binding pocket (Walker, supra; Wilson, supra).
Because of the apparently harmful role of IL-1 in many disease conditions, therapeutic strategies aimed at reducing the production or action of IL-1 have been proposed. For example, one approach by which to inhibit matIL-1xcex1 production and secretion would be to block the activity of ICE with a specific ICE inhibitor. The ability to produce active ICE in vitro is therefore highly desirable to allow for the study of its structure and function. However, in vitro production of ICE can be hampered by the instability of the protein, in particular as a result of autocatalytic degradation which leads to inactive protein.
This invention provides modified human ICE proteins that retain the proteolytic activity of unmodified human ICE and that exhibit increased stability in vitro compared to modified ICE. One aspect of the invention pertains to a modified form of ICE comprising an amino acid sequence wherein an amino acid corresponding to aspartic acid at position 381 of unmodified ICE (SEQ ID NO: 2) is replaced with a mutant amino acid structure. This mutant amino acid structure is one that is capable of forming a salt bridge with an amino acid corresponding to arginine at position 383 of unmodified ICE, such that the modified ICE exhibits proteolytic activity and has increased stability compared to unmodified ICE. The mutant amino acid structure that replaces the amino acid corresponding to Asp381 can be a natural amino acid or a non-natural amino acid. In the most preferred embodiment, the mutant amino acid structure is a glutamic acid residue. In other embodiments, the mutant amino acid structure is selected from the group consisting of serine, threonine, asparagine and glutamine.
Another aspect of the invention pertains to nucleic acid molecules encoding a modified p10 subunit of ICE. The modified p10 subunit encoded by the nucleic acid molecule comprises an amino acid sequence wherein an amino acid corresponding to aspartic acid at position 381 of unmodified ICE (SEQ ID NO: 2) is replaced with a mutant amino acid structure. The mutant amino acid structure is one that is capable of forming a salt bridge with an amino acid corresponding to arginine at position 383 of unmodified ICE, such that the modified p10 subunit associates with a p20 subunit to form a modified ICE that retains proteolytic activity and exhibits increased stability compared to unmodified ICE. In the most preferred embodiment, the p10 subunit encoded by the nucleic acid molecule has a glutamic acid residue at the position corresponding to position 381 of unmodified ICE. In other embodiments, the p10 subunit has a serine, threonine, asparagine or glutamine residue at the position corresponding to position 381 of unmodified ICE.
A nucleic acid molecule of the invention encoding a modified p10 subunit of ICE can be incorporated into a recombinant expression vector. In one embodiment, the recombinant expression vector encodes the modified p10 subunit of ICE (i. e., about amino acids 317 to 404). In another embodiment, the recombinant expression vector encodes the modified p10 subunit of ICE and also encodes the p20 subunit of ICE (i.e., about amino acids 120-197). For example, in one embodiment, the recombinant expression vector encodes the p30 form of ICE, comprising about amino acids 120-404. This p30 form of ICE undergoes maturational processing to produce a modified p10 subunit and a p20 subunit.
The recombinant expression vectors of the invention can be introduced into host cells to produce modified ICE proteins. In one embodiment, a modified p10 subunit is expressed recombinantly in a host cell. denatured and refolded with a p20 subunit of ICE to form a modified ICE protein of the invention. In another embodiment, a modified p10 subunit is coexpressed with a p20 subunit in the same host cell (using either two separate expression vectors or one expression vector encoding both the p10 and p20 subunits, such as a vector encoding p30), thereby producing a modified ICE protein of the invention.
The modified ICE proteins of the invention are cysteine proteases that exhibit proteolytic activity against ICE substrates. Accordingly, a modified ICE protein of the invention can be used to cleave an ICE substrate by contacting the ICE substrate with the modified ICE such that the ICE substrate is cleaved. Moreover, the modified ICE proteins of the invention can be used in screening assays to identify modulators (e.g., inhibitors or stimulators) of ICE protease activity. Still further, the enhanced stability of the modified ICE proteins of the invention makes them particularly well-suited for the preparation of crystalline ICE for use in X-ray cystallographic analysis (e.g., for structure-based design of ICE inhibitors).